The initiation of immune responses within lymphoid tissue involves a carefully orchestrated movement of activated lymphocytes within an elaborate network of stationary stromal cells. Lymphoid tissue architecture is comprised of distinguishable microanatomic neighborhoods that are based on a complex arrangement of stromal cells. Due to the secretion of distinctive combinations of chemokines by stromal cell subsets, the chemokine fingerprint within B cell follicles is distinguishable from its neighboring T cell zone. Through shifts in chemokine receptor expression after activation, antigen engaged B and T cells reposition themselves to congregate within new niches, promoting their cognate encounters and enabling key steps in the differentiation of critical effectors. Although on a macro scale, the uniform distribution of nave B cells throughout the follicle might suggest a similar uniformity in the stationary structural underpinnings, there are instead many stromal cell subsets positioned within follicles. In addition to the well characterized follicular dendritic cell (FDC), B cell folicles also harbor lymph and blood endothelial cells, the later covered by pericytes. Fibroblastic reticular cells rim the inside edge of the border of the follicle closest to the T cell zone, wheres the subcapsular sinus lining is a collection of yet more cell types including a marginal reticular cell with descending dendrites. Despite the critical role of stromal cells in defining the landmark for lymphocyte orientation during initial activation events, many fundamental aspects of B cell interactions with follicular stroma are unknown. Here we propose to examine the interactions between B cells and the stromal cell subsets that make up the follicular infrastructure. Aim 1: Define the stromal cell framework of B cell follicles. Using intravital microscopy of fluorescent bone marrow chimeras, the relative positioning of blood and lymph vessels vis--vis FDCs and other intra-follicular stromal cells will be defined. We will assess the consistency of their juxtapositions, spacing and associated cell types before and after immunization. Aim 2: Define the propensity of nave and activated B cells to interact with stromal cell subsets via intravital microscopy. The morphodynamics and mode of propulsion between B cells in contact with stroma will be compared to those within regions that lack stroma, and the impact of blood or lymph-borne antigen/adjuvants on this behavior determined. Motility parameters of nave and activated B cells within stromal gaps will be quantified to test the hypothesis that such movement is transient and a directed shift from one stromal contact to another. Aim 3: Examine B cells in situ for intracellular indicators of potential signaling events during contacts with strmal cell subsets. B cells with synaptic morphology adjacent to stromal cells will be examined by histology for intracellular indicators of Notch, BMP and BCR mediated signaling coincident with stromal cell subset contacts. To test the hypothesis that such contacts support BCR derived tonic signaling, we will perform intravital imaging of B cells transfected with a NFAT-eGFP retroviral construct within LNs of fluorescent BM chimeras.